CHARACTERIZATION OF TWO NOVEL MARINE CALICIVIRUSES: MOLECULAR AND SEROLOGICAL APPROACHES FOR IMPROVED DIAGNOSTICS By SHASTA DAWN McCLENAHAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1 © 2008 Shasta Dawn McClenahan 2 To my best friend and husband, Justin McClenahan. He has supported me unconditionally over the past nine years to help me get to the place I am today. He has followed me wherever I wanted to go for my education and now it is my turn to follow his lead. 3 ACKNOWLEDGMENTS I acknowledge the support from my mentor Dr. Carlos Romero. Without his acceptance into this program, I would not be here today. His guidance and mentoring over the past four years were extremely valuable during my doctorate degree, and I am forever indebted to him. I acknowledge my committee members, Dr. Ayalew Mergia, Dr. Peter McGuire, Dr. James Maruniak, and Dr. Charles Manire. Their assistance and guidance helped me tremendously during my research and preparation of this dissertation. I also acknowledge Dr. Kim Green from the National Institutes of Health, and all of her staff, especially Dr. Karin Bok and Dr. Slava Sosnovtsev. They welcomed me into their lab with open arms, spent much of their valuable time with me, and taught me so much. Their experience with baculovirus expression was valuable, and helped with a crucial part of my research. I also thank Dr. Kathy Burek, with Alaska Veterinary Pathology Services, and Dr. Kimberlee Beckmen with the Alaska Department of Fish and Game. They made the collection of Steller sea lion samples possible, and helped me tremendously throughout this project. I thank my husband, and number one fan, Justin McClenahan. He has been there for me more than any other person to offer support. I also thank my family including my parents, Don and Kathi Cooper, and my sisters Shiloh Cooper and Sarah Cooper. I also thank Rebecca Grant, my partner in crime for the last four years of this program. She has been there to offer help and useful suggestions during difficult experiments, but mostly for just being a good friend. I also thank Heather Townsend. Her friendship and help navigating the University of Florida, the Veterinary School, and the Marine Mammal Program were very valuable. She especially made our immunology course more fun, and easier to get through, and she makes me laugh more than anyone else that I know. 4 I acknowledge the grants and funding that made this research possible. This research was funded by sub agreement No. 6402-181-L0-C between the University of Florida and the Center for Biological Defense, University of South Florida, under contract No. DAAD13-01-C-0043 of the Office of Naval Research. I would also like to thank the Marine Mammal Health Program for funding my research program, and the Department of Infectious Diseases and Pathology of the College of Veterinary Medicine, University of Florida. This research would also not be possible if it were not for the many permits and agencies that allowed this research to be conducted. Tissues, lesions, and swabs from Steller sea lions were harvested under permit numbers 358-1564-03 and 782-1889 from the National Oceanic and Atmospheric administration, US Department of Commerce. The University of Florida’s Aquatic Animal Health permits from the National Marine Fisheries permit numbers 1054-1731-00 and 1054-1731-01. Institutional Animal Care and Use Committee (IUCAC) numbers for this research were D438, D805, E853, and E883. The stranding networks in the Southeastern United States also made tissue samples available for research. Some of the organizations and individuals in this network included Mote Marine Lab, Dr. Ruth Ewing and the National Marine Fisheries Service, Dr. Connie Chevis, Florida Fish and Wildlife Conservation Commission, Bob Bonde and the United States Geological Survey, and the University of Florida stranding response team, veterinarians, pathologists, and necropsy teams. I would also like to thank several individuals from the Pacific Coast for marine mammal samples including Dr. Pam Tuomi and Dr. Carol Stephens of the Alaska Sea Life Center, Dr. Judy St. Leger of SeaWorld San Diego, and Dr. Tracey Goldstein of The Marine Mammal Center. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES.........................................................................................................................10 LIST OF FIGURES .......................................................................................................................12 LIST OF ABBREVIATIONS........................................................................................................14 ABSTRACT...................................................................................................................................17 CHAPTER 1 INTRODUCTION ..................................................................................................................19 Caliciviridae............................................................................................................................19 Phylogeny........................................................................................................................19 Genomic Organization.....................................................................................................20 Capsid Structure ..............................................................................................................21 Viral Replication .............................................................................................................22 Viral Members of the Caliciviridae........................................................................................24 Norovirus Genus..............................................................................................................24 Sapovirus Genus..............................................................................................................26 Lagovirus Genus..............................................................................................................26 Vesivirus Genus...............................................................................................................27 Vesivirus History....................................................................................................................28 Vesicular Exanthema of Swine .......................................................................................28 Marine Vesiviruses..........................................................................................................30 Marine Vesivirus Link to Vesicular Exanthema of Swine..............................................31 Other Terrestrial Vesiviruses of an Ocean Origin...........................................................32 Caliciviridae Pathogenesis......................................................................................................35 Vesivirus Receptor ..........................................................................................................37 Immune Response to Caliciviruses .................................................................................38 Epidemiology of Caliciviruses ...............................................................................................39 Viral Transmission ..........................................................................................................39 Viral Prevalence ..............................................................................................................40 Control and Prevention of Calicivirus Infections............................................................42 Viral Evolution and Quasispecies...........................................................................................43 Steller Sea Lion Decline .........................................................................................................44 2 GENOMIC CHARACTERIZATION OF NOVEL MARINE VESIVIRUSES FROM STELLER SEA LIONS (Eumetopias jubatus) FROM ALASKA.........................................51 Introduction.............................................................................................................................51 Materials and Methods ...........................................................................................................53 6 Source of Viruses ............................................................................................................53 Virus Isolation in Cell Culture ........................................................................................53 Electron Microscopy .......................................................................................................54 Extraction of RNA from Cell Cultures............................................................................54 Reverse Transcription......................................................................................................55 Oligonucleotide Primers..................................................................................................55 Polymerase Chain Reaction.............................................................................................55 Capsid gene fragment...............................................................................................55